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Agrobacterium harboring a Ti or a Ri plasmid can efficiently transfer a portion of these plasmids, the T-DNA, into plant cells. Transfer of the T-DNA into the plant cell is induced by signal compounds present at the site of a plant wound and requires T-DNA border sequences at both ends of the T-DNA and trans-acting virulence gene products (vir) encoded by the Ti or Ri plasmid. The transferred T-DNA is then targeted to the nucleus and integrated into the plant genome.
Light-dependent short hypocotyls (LSH) proteins found in plants are known to mediate light regulation in seedling development, meristem maintenance, and shoot regeneration. There are currently ten known family members of this protein family, and structurally they contain a central domain of unknown function (DUF640) flanked by amino- and carboxy-terminal variable domains. LSH proteins also contain putative monopartite SV40-type NLS sequences overlapping the DUF640 and the adjacent carboxy-terminal variable domain.
A number of techniques exist for introducing exogenous DNA into plant cells, such as protoplasts, which are capable of subsequent regeneration, such as, microinjection of naked DNA, electroporation, Ca/PEG precipitation, and particle bombardment-mediated delivery, so called “biolistics.”
A common previously utilized technique for the genetic engineering of plants involves the use of the soil-dwelling plant pathogenic bacterium Agrobacterium. Agrobacterium has the natural ability to transfer a portion of its DNA, referred to as T-DNA, into the genome of susceptible plant cells. By changing the native T-DNA in an Agrobacterium strain, it is possible to use this unique trait of Agrobacterium to transfer desired genes into single plant cells. While Agrobacterium are typically restricted to infecting dicotyledonous species under natural conditions, by manipulating the conditions of infection, efficient transformation of monocots, including some crop species has been possible. Common to the methods specified above is the integration of the exogenous DNA into a random site in the plant chromosome. While useful for many applications, random integration of transgenes leaves a number of difficulties. For example, the targeted disruption of an endogenous gene requires that integration occur at a specified locus in the host plant genome. Gene targeting is difficult when using the Agrobacterium method of transformation, because an Agrobacterium protein called VirE2 masks the naked T-DNA when transfected into the host plant cell, which blocks any chance of site directed genetic modification by homologous recombination.
Currently, there is not an elegant solution to transform cells with T-DNA at a site of interest in the host genome.